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Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology.

Cell cortices are responsible for the resilience and morphological dynamics of cells. Measuring their mechanical properties is impeded by contributions from other filament types, organelles, and the crowded cytoplasm. We established a versatile concept for the precise assessment of cortical viscoelasticity based on force cycle experiments paired with continuum mechanics. Apical cell membranes of confluent MDCK II cells were deposited on porous substrates and locally deformed. Force cycles could be described with a time-dependent area compressibility modulus obeying the same power law as employed for whole cells. The reduced fluidity of apical cell membranes compared to living cells could partially be restored by reactivating myosin motors. A comparison with artificial minimal actin cortices (MACs) reveals lower stiffness and higher fluidity attributed to missing cross-links in MACs.

The mechanical response of adherent, polarized cells to indentation is frequently attributed to the presence of an endogenous actin cortex attached to the inner leaflet of the plasma membrane. Here, we scrutinized the elastic properties of apical membranes separated from living cells and attached to a porous mesh in the absence of intracellular factors originating from the cytosol, organelles, the substrate, neighbors, and the nucleus. We found that a tension-based model describes the data very well providing essentially the prestress of the shell generated by adhesion of the apical membrane patches to the pore rim and the apparent area compressibility modulus, an intrinsic elastic modulus modulated by the surface excess stored in membrane reservoirs. Removal of membrane-associated proteins by proteases decreases the area compressibility modulus, whereas fixation and cross-linking of proteins with glutaraldehyde increases it.

Mechanical phenotyping of adherent cells has become a serious tool in cell biology to understand how cells respond to their environment and eventually to identify disease patterns such as the malignancy of cancer cells. In the steady state, homeostasis is of pivotal importance, and cells strive to maintain their internal stresses even in challenging environments and in response to external chemical and mechanical stimuli. However, a major problem exists in determining mechanical properties because many techniques, such as atomic force microscopy, that assess these properties of adherent cells locally can only address a limited number of cells and provide elastic moduli that vary substantially from cell to cell. The origin of this spread in stiffness values is largely unknown and might limit the significance of measurements. Possible reasons for the disparity are variations in cell shape and size, as well as biological reasons such as the cell cycle or polarization state of the cell. Here, we show that stiffness of adherent epithelial cells rises with increasing projected apical cell area in a nonlinear fashion. This size stiffening not only occurs as a consequence of varying cell-seeding densities, it can also be observed within a small area of a particular cell culture. Experiments with single adherent cells attached to defined areas via microcontact printing show that size stiffening is limited to cells of a confluent monolayer. This leads to the conclusion that cells possibly regulate their size distribution through cortical stress, which is enhanced in larger cells and reduced in smaller cells.

Viscoelastic properties of epithelial cells subject to shape changes were monitored by indentation-retraction/relaxation experiments. MDCK II cells cultured on extensible polydimethylsiloxane substrates were laterally stretched and, in response, displayed increased cortex contractility and loss of excess surface area. Thereby, the cells preserve their fluidity but inevitably become stiffer. We found similar behavior in demixed cell monolayers of ZO-1/2 double knock down (dKD) cells, cells exposed to different temperatures and after removal of cholesterol from the plasma membrane. Conversely, the mechanical response of single cells adhered onto differently sized patches displays no visible rheological change. Sacrificing excess surface area allows the cells to respond to mechanical challenges without losing their ability to flow. They gain a new degree of freedom that permits resolving the interdependence of fluidity β on stiffness $${K}_{{{{{{{{\rm{A}}}}}}}}}^{0}$$ K A 0 . We also propose a model that permits to tell apart contributions from excess membrane area and excess cell surface area.

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